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Prevention of restenosis with intravascular beta-radiotherapy.

Beta radiation has been clearly shown, in a specific dose range, to be highly effective in the inhibition of the restenotic process after balloon or stent injury in animal experiments, as well as in randomized, placebo-controlled human trials. The major advantage of beta radiation, in comparison with gamma radiation, is a significantly lower radiation exposure to the personnel and patient, and easier adaptability to existing cardiac catheterization laboratories. Rapidly accumulating evidence indicates that the two major problems, late thrombosis and edge stenosis, may be minimized with prolonged antiplatelet therapy (6 months or more) and broader radiation coverage of the intervention site. Although there may be better, safer, and easier options to reduce restenosis in the years to come, intravascular radiotherapy is the first breakthrough modality that has been shown to significantly reduce restenosis after percutaneous vascular interventions.

Angioplasty↗

Bacterial corneoscleritis complicating pterygium excision.

Pterygium excision was complicated by bacterial corneoscleritis and endophthalmitis in two patients, one who had received postoperative beta irradiation and the other topical thiotepa. These complications followed surgery by two and six weeks. Causative organisms were Streptococcus pneumoniae and Pseudomonas aeruginosa, respectively. Despite aggressive therapy, visual outcome was poor in both cases. These cases demonstrate that, although infrequent, complications of pterygium excision can be destructive and visually disabling.

Adult↗

Biosynthetic labeling with 32P: radiation damage to mammalian cells.

Theoretical calculations showed that biosynthetic radiolabeling of cells using typical concentrations of 32P (1 mCi/ml) resulted in high radiation doses (200-500 rad/h) being absorbed by the cells. Subsequent investigations with a mouse myelomonocytic leukemia cell line (WEHI-3B(D+)) showed significant loss of replicative ability during brief (less than 1 h) exposures to 1 mCi/ml of 32P. Complete loss of cell replicative ability was found with isotopic doses less than 100 rad (i.e., 100 muCi/ml for 5 h). Experiments employing a less radiosensitive pre-B-cell line (18.81) revealed that significant loss of viability occurred during incubation with 32P under identical conditions to those employed for the WEHI-3B(D+) cell line. Control experiments utilizing decayed batches of 32P and physical separation of the isotope solution from the cells confirmed that the cytotoxicity was caused by radiation emission rather than the presence of toxic components in the isotopic solution. The radiation doses absorbed by cells biosynthetically labeled with 59Fe, 33P, 35S, and 14C were calculated. Although significant levels of radiation can be absorbed 32P was considerably more radiotoxic than the other isotopes. The results of calculations indicated that the judicious choice of container geometry could reduce the absorbed radiation dose from 32P solutions. In particular the biosynthetic radiolabeling of cells in capillary tubes (diameter less than 1 mm) can reduce the absorbed rate to less than one-tenth of the dose received by cells suspended in Petri dishes or centrifuge tubes.

Animals↗

Application of simultaneous determination of 3H, 14C, and 22Na by liquid scintillation counting to the measurement of cellular ion-transport.

A liquid scintillation counting method for simultaneous determination of three radioactive nuclides (3H, 14C, and 22Na) of biological interest was studied. By comparing the beta spectra of the three nuclides, their counting energy ranges, A, B, and C, were determined. 22NA was set high enough to avoid any spillover counts from lower-energy nuclides. Region A for 3H was set to maximize the counting efficiency. A good correlation between the counting efficiency for 22Na in region C and the counting efficiency of other nuclides in all regions was obtained. Prior to 3H and 14C dpm calculations, the 22Na counts spilled down in regions A and B were subtracted from the total counts in regions A and B. A simple linear equation was then used to compute 3H and 14C dpm. Findings show that the method presented is adaptable for highly quenched samples up to quenching indices of tSIE = 100. The method is useful for studying the biological transport coupled to Na+.

Beta Particles↗

Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apo A-I-containing subpopulations.

Two-dimensional electrophoresis has been used to resolve 12 distinct apo A-I-containing high-density lipoprotein (HDL) subpopulations in human plasma. The subpopulations were quantitated by 125I-labeled, monospecific antibody and phosphor-imaging. Modification and standardization of the agarose electrophoresis (first dimension) enabled us to recognize new HDL subpopulations. Lipoprotein mobilities in agarose were expressed relative to the mobility of the sample's endogenous albumin. We demonstrated the presence of lipoproteins with mobilities faster than and similar to albumin, as well as subpopulations with mobilities slower than albumin. We refer to these as pre alpha, alpha and pre beta, respectively. Lipoprotein molecular sizes were determined with a non-denaturing polyacrylamide gradient gel electrophoresis (PAGE) (2% to 36%) in the second dimension. Internal standard of 125I-labeled proteins of known molecular size was run simultaneously in each gel permitting accurate size determination. We have demonstrated that ultracentrifugally-isolated lipoproteins are different from the native apo A-I-containing subpopulations. The major difference observed was the loss of pre beta 1 and pre beta 2 particles from the d < 1.21 g/ml fractions to the d > 1.21 g/ml fractions. Possible physiologic and pathologic implications of these findings are also discussed.

Albumins↗

Radiation inactivation analysis of kidney microvillar peptidases.

Five membrane peptidases were studied by radiation inactivation analysis of pig kidney microvillar membranes. One heterodimeric enzyme, gamma-glutamyl transferase, presented a target size corresponding to the dimeric Mr. The other enzymes are known to be homodimers. Three of these, aminopeptidase A. aminopeptidase N and dipeptidyl peptidase IV, gave results clearly indicating the monomer to be the target and, hence, in this group the association of the subunits was not essential for activity. The target size for endopeptidase-24.11 was intermediate between those for monomer and dimer and its functional state was not resolved by the experiments.

Animals↗

Lipoproteins do not influence cholesterol synthesis in freshly isolated rat hepatocytes, cautionary note.

1. Suspensions of freshly isolated rat hepatocytes were used to study the effects of native and derivatized lipoproteins on the rate of cholesterogenesis. 2. Short-term incubation of the hepatocytes with a variety of lipoproteins failed to modify the rate of cholesterol synthesis as determined by the incorporation of tritium from tritiated water into cholesterol after separation from other lipids by thin-layer chromatography. 3. Neither an increase in the cholesterol content of the particles (beta-very-low-density lipoproteins) nor derivatization of the lipoproteins (lactosylated-low-density lipoproteins or high-density lipoproteins associated with a tris-galactoside-terminated cholesterol derivative) nor cholesterol-containing liposomes were effective in this respect. 4. Whether this behaviour represents an artefact of the isolated hepatocyte preparation is unknown yet.

Animals↗

A study of the lipid transport system in the cat, Felix domesticus.

Feline serum lipoproteins were fractionated into four distinct classes by density gradient ultracentrifugation and characterized with respect to physical and chemical properties. The distribution of serum lipids, lipoproteins and apolipoproteins was quite unlike that in man, the cat having five times as much high density lipoproteins (HDL) as low density lipoproteins (LDL). The lipoproteins in the d less than 1.019 g/ml fraction of cats were larger and were richer in triglycerides than their human counterparts and contained a considerable amount of beta-migrating particles. The low density lipoproteins of cats and man had similar chemical composition, but cat LDL had a higher negative charge, were smaller and contained apoprotein A-I. Cat HDL consisted of two distinct subfractions HDL2 and HDL3 with similar density boundaries and particle size as in man. In cat serum and HDL fraction apoprotein A-II was a minor component. Like human serum, fasting cat serum contained only the larger species of apoprotein B, apo B-100, whereas intestinal lymph contained exclusively the smaller apo B-48. Post heparin feline and human plasma possessed both lipoprotein lipase and hepatic lipase. Chylomicrons formed after a fat load in cats were removed from the circulation as rapidly as in man. It is concluded, that the cat is another animal model of potential interest for the study of lipoprotein metabolism.

Animals↗

Effect of endothelium on beta-VLDL metabolism by cultured smooth muscle cells of differing phenotype.

The effect of endothelial cells (EC) on the binding and internalisation of beta-very low density lipoprotein (beta-VLDL) and the subsequent accumulation of lipid was investigated in cultured smooth muscle cells (SMC) of different phenotype. The following combinations were examined: (i) SMC cultured and incubated with 125I-beta-VLDL without EC: "control" cultures; (ii) SMC co-cultured with EC and incubated with 125I-beta-VLDL without EC: "separated" cultures; and (iii) SMC co-cultured with EC and incubated with 125I-beta-VLDL in the presence of EC: "co-incubated" cultures. SMC were in the contractile (CON), reversible synthetic (RS) or irreversible synthetic (IRS) phenotype and EC were either actively proliferating or confluent and quiescent. All three SMC phenotypes showed the greatest capacity to bind and internalise 125I-beta-VLDL with accumulation of lipid when "co-incubated" with confluent EC. SMC "co-incubated" with proliferating EC showed a lower capacity to bind and internalise the lipoprotein and accumulate lipid, while "control" SMC showed the lowest capacity for all phenotypes. IRS SMC bound more 125I-beta-VLDL than either RS or CON state phenotypes. In addition, IRS SMC "co-incubated" with confluent EC showed the greatest degree of binding, and IRS SMC incubated with EC-conditioned medium and EC-conditioned 125I-beta-VLDL showed a significant increase in binding above control (fresh medium and fresh 125I-beta-VLDL). The degree of binding 125I-beta-VLDL to SMC was affected by the functional state of the EC. That is, SMC "co-incubated" with confluent EC bound more lipoprotein than SMC "co-incubated" with the same number of proliferating EC. These results are consistent with observations by others who report preferential lipid accumulation in regions of denuded artery recently recovered by endothelium compared with regions lacking an endothelium. The results also indicate that the EC both modify the beta-VLDL particle and affect the biology of the SMC themselves.

Animals↗

Direct counting of tritium by dissolving Microelisa wells in scintillation fluid.

Radioassays with low-energy beta-emitting nuclides (e.g., 3H, 14C, 35S) in 96-well plastic plates are tedious and frequently inaccurate because of the necessity of quantitatively removing and transferring the contents of each well to scintillation fluid. We therefore investigated the possibility of counting these nuclides by directly placing the entire break-apart well (Microelisa) along with the sample into one of four scintillation counting fluids: toluene-PPO-POPOP with or without Protosol, ACSR, and EcoLite. Although some of these scintillation fluids fully dissolved the plastic wells and other did not, we found that the presence of the wells did not appreciably interfere with the efficiency of tritium counting. This technique saves considerable time and reduces possible errors in liquid scintillation counting of samples from plastic microtitration plates.

Beta Particles↗

Lymphocyte proliferation and cytotoxic assays using flat-bed scintillation counting.

Lymphocyte 51Cr release and [3H]thymidine uptake assays were evaluated with respect to measurement of sample radioactivity using the flat-bed scintillation counter. 51Cr lysates were spotted onto a glass fibre filter sheet while [3H]thymidine-labelled cells were filtered onto a similar sheet using a cell harvester. The 96 samples were rapidly processed for counting, without removal of individual sample areas. Either form of preparation showed good linearity of count rate with the quantity of material on the filter. Reproducibility was good; the coefficient of variation for 96 samples being within 5%. The low background and high efficiency of this counter results in increased assay sensitivity and allows considerable economies in materials to be made. A commercial version of the counter has six counting heads permitting a high rate of sample throughput.

Beta Particles↗